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Glutamine independence is a selectable feature of pluripotent stem cells

Nature Metabolism volume 1, pages 676–687 (2019)Cite this article

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Abstract

Most rapidly proliferating mammalian cells rely on the oxidation of exogenous glutamine to support cell proliferation. We previously found that culture of mouse embryonic stem cells in the presence of inhibitors against mitogen-activated protein kinase kinase and glycogen synthase kinase 3 beta to maintain pluripotency reduces cellular reliance on glutamine for tricarboxylic acid cycle anaplerosis, enabling embryonic stem cells to proliferate in the absence of exogenous glutamine. Here we show that reduced dependence on exogenous glutamine is a generalizable feature of pluripotent stem cells. Enhancing self-renewal, through either overexpression of pluripotency-associated transcription factors or altered signal transduction, decreases the use of glutamine-derived carbons in the tricarboxylic acid cycle. As a result, cells with the highest potential for self-renewal can be enriched by transient culture in glutamine-deficient media. During pluripotent cell culture or reprogramming to pluripotency, transient glutamine withdrawal selectively leads to the elimination of non-pluripotent cells. These data reveal that reduced dependence on glutamine anaplerosis is an inherent feature of self-renewing pluripotent stem cells and reveal a simple, non-invasive mechanism to select for mouse and human pluripotent stem cells within a heterogeneous population during both embryonic stem cell passage and induced pluripotent cell reprogramming.

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Fig. 1: Glutamine anaplerosis is reduced in ESCs with enhanced self-renewal.
Fig. 2: Enhanced self-renewal improves glutamine-independent survival.
Fig. 3: Transient glutamine withdrawal enhances ESC self-renewal.
Fig. 4: Transient glutamine withdrawal improves mouse somatic cell reprogramming to pluripotency and enhances human ESC self-renewal.

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Data availability

The data that support the findings of this study are available from the corresponding author upon reasonable request. Source data for all gas-chromatography–mass spectrometry data are provided in Supplementary Table 1.

Code availability

The MATLAB code that supports the findings of this study is also available from the corresponding author upon reasonable request.

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Acknowledgements

We thank the Finley laboratory for discussion and A. Intlekofer for critical feedback. We thank A. Smith (University of Cambridge) for the gift of the chimeric LIF receptor and R. Jaenisch (Whitehead Institute for Biomedical Research) for the gift of the Nanog-GFP ESCs. S.A.V. is a Parker Fellow with the Parker Institute of Cancer Immunotherapy. B.P.R. was supported by an National Institutes of Health (NIH) T32 Training Grant in Molecular and Cellular Biology (no. T32GM008539). L.W.S.F. is a Searle Scholar and was a Dale F. Frey-William Raveis Charitable Fund Scientist supported by the Damon Runyon Cancer Research Foundation (no. DFS-23-17). This work was additionally supported by the Concern Foundation and the Anna Fuller Fund (to L.W.S.F.), The Starr Foundation (no. I11-0039 to L.W.S.F.), a Pathway to Independence Award from the NIH (no. R00 CA191021 to C.C.-F.), NIH/National Institute of Diabetes and Digestive and Kidney Diseases (no. R01DK096239 to D.H.) and the Memorial Sloan Kettering Cancer Center Support Grant no. P30 CA008748.

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Authors and Affiliations

  1. Cancer Biology and Genetics Program, Memorial Sloan Kettering Cancer Center, New York, NY, USA

    Santosha A. Vardhana & Craig B. Thompson

  2. Center for Epigenetics Research, Memorial Sloan Kettering Cancer Center, New York, NY, USA

    Santosha A. Vardhana, Paige K. Arnold, Yanyang Chen, Craig B. Thompson & Lydia W. S. Finley

  3. Cell Biology Program, Memorial Sloan Kettering Cancer Center, New York, NY, USA

    Paige K. Arnold, Yanyang Chen & Lydia W. S. Finley

  4. Louis V. Gerstner, Jr., Graduate School of Biomedical Sciences, Memorial Sloan Kettering Cancer Center, New York, NY, USA

    Paige K. Arnold

  5. Developmental Biology Program, Memorial Sloan Kettering Cancer Center, New York, NY, USA

    Bess P. Rosen & Danwei Huangfu

  6. Laboratory of Chromatin Biology and Epigenetics, The Rockefeller University, New York, NY, USA

    Bryce W. Carey

  7. Center for Genomics & Systems Biology, Department of Biology, New York University, New York, NY, USA

    Carlos Carmona-Fontaine

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Contributions

S.A.V. and L.W.S.F. conceived the study. S.A.V., P.K.A and L.W.S.F. performed all the experiments with assistance from Y.C. B.W.C. assisted with the reprogramming experiments. B.P.R. and D.H. performed the human ESC experiments. C.C.-F. performed the immunofluorescence and image analysis. C.B.T. provided additional work in study conception and guidance. S.A.V. and L.W.S.F. wrote the manuscript.

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Correspondence to Lydia W. S. Finley.

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Competing interests

C.B.T. is a founder of Agios Pharmaceuticals and a member of its scientific advisory board. He also previously served on the board of directors of Merck and Charles River Laboratories.

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Supplementary Information

Supplementary Figs. 1–6

Reporting Summary

Supplementary Table 1

Source data for all gas chromatography–mass spectrometry experiments

Supplementary Table 2

Calculation of reprogramming efficiency, related to Fig. 4c

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Vardhana, S.A., Arnold, P.K., Rosen, B.P. et al. Glutamine independence is a selectable feature of pluripotent stem cells. Nat Metab 1, 676–687 (2019). https://doi.org/10.1038/s42255-019-0082-3

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